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Lecture 4

Lecture 4. Mosaic analysis Maternal/zygotic screens Non-complementation screen Cytogenomics to genomics Designer deletions Gene disruption in Drosophila Systematic RNAi screens. Lab 4 schedule. MONDAY SECTION: Sunday 9:30-11:30 AM come in to start your in situ. WSC 339

lillian-griffith
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Lecture 4

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  1. Lecture 4 • Mosaic analysis • Maternal/zygotic screens • Non-complementation screen • Cytogenomics to genomics • Designer deletions • Gene disruption in Drosophila • Systematic RNAi screens

  2. Lab 4 schedule • MONDAY SECTION: Sunday 9:30-11:30 AM come in to start your in situ. WSC 339 • TUESDAY SECTION: Monday 10-12AM come in to start your in situ. B&G 3015 • On Monday and Tuesday the lab B&G 3015 will be open at 10AM to start your washes.

  3. Biology 4540g Midterm Examination Tuesday February 28, 2012 Room UCC 63 7-10 PM

  4. Mosaic Analysis What is a genetic mosaic?

  5. Mitotic recombination - w- - + w+ +

  6. Mitotic recombination - w- - + w+ +

  7. Mitotic recombination - - + +

  8. Mitotic recombination w- w- - - + + w+ w+

  9. Mitotic recombination w- Eye w- - - + + w+ w- w+ w+

  10. Mitotic recombination Clones of cells X-rays Indiscriminant crossing over

  11. Flip mediated site specific recombination Yeast two micron plasmid Flip A C D A FRT FRT FRT B FRT B D C

  12. Flip mediated mitotic recombination FRT FRT

  13. Flip mediated mitotic recombination + FRT Sb FRT

  14. Flip mediated mitotic recombination + + FRT FRT Sb FRT Sb FRT

  15. Flip mediated mitotic recombination hspFLP + + FRT FRT Sb FRT Sb FRT

  16. Flip mediated mitotic recombination hspFLP + + FRT FRT Sb FLP FRT Sb FRT

  17. Flip mediated mitotic recombination hspFLP + + FRT FRT Sb FLP FRT Sb FRT

  18. Flip mediated mitotic recombination + + FRT FRT Sb FRT Sb FRT

  19. Flip mediated mitotic recombination + FRT + FRT Sb FRT Sb FRT

  20. Flip mediated mitotic recombination + FRT + FRT Sb FRT Sb FRT

  21. Mosaic analysis • Assay adult phenotype of an embryonic lethal. • Determine the site of action of a gene product. • Analyze Zygotic lethal/Maternal effect mutants.

  22. Pure maternal and zygotic screens + Bal Oogenesis Zygote + Bal

  23. Pure zygotic screens + Bal Oogenesis Zygote Assume pure expression of a gene from the zygotic genome only. + Bal

  24. Pure maternal screens + Bal Oogenesis Zygote Assume pure maternal expression of a gene + Bal

  25. Exceptions to the strict assumptions 1. Maternal rescue 2. Zygotic lethal/Maternal effect

  26. Maternal Rescue Zygote + Bal The maternally supplied transcript rescues the zygotic loss of function. Oogenesis

  27. Maternal Rescue Hide an essential role for a gene in embryonic pattern formation. Pure zygotic screens may miss important functions.

  28. Pure maternal screens + Bal Oogenesis Zygote Assume pure maternal expression of a gene + Bal

  29. Zygotic lethal/Maternal effect + Bal Oogenesis Zygote Dies at late larval stage or pupal stage + Bal We can not assay if the gene has a maternal effect phenotype.

  30. Zygotic lethal/Maternal effect Since a homozygous adult mother can not be established, we can not assay whether the gene contributes information to the egg.

  31. Screen for maternal rescue and zygotic lethal/maternal effect 1/2 to 2/3 of lethal mutations are not embyonic lethal. Norbert Perrimon collected a large # of late larval and pupal lethals.

  32. Properties of OvoD OvoD dominant female sterile germ line specific cell autonomous OvoD No eggs

  33. Mosaic analysis with OvoD OvoD + No eggs l + Irradiate with X-rays

  34. Mosaic analysis OvoD + l +

  35. Mosaic analysis OvoD + l +

  36. Mosaic analysis OvoD + l +

  37. Mosaic analysis OvoD + OvoD + OvoD + l + l + l +

  38. Mosaic analysis OvoD + OvoD + l + Egg maker l +

  39. Maternal rescue vs. Maternal effect Maternal rescue can be rescued by the paternal allele. Maternal effect is independent of the genotype of the sperm.

  40. Maternal effect Egg chamber l l l +

  41. Maternal effect l Embryonic phenotype l l Embryonic phenotype +

  42. Maternal rescue Egg chamber l l l +

  43. Maternal rescue l Embryonic phenotype l l No phenotype rescued by the paternal allele +

  44. Non-complementation screen Isolation of more independent mutant alleles in a gene.

  45. Non-complementation screen wild-type en1/en1

  46. Non-complementation screen en1/en1engrailed phenotype en1/+wild-type phenotype en1/deletion engrailed phenotype en1/enx

  47. Non-complementation screen EMS F0 CyO/L virgin females X b pr cn males F1 en1/en1virgin females X b pr cn*/CyO 4,000 crosses with individual males F2 Screen vials for flies with the en phenotype

  48. Vials with no engrailed flies 50% 50% b pr en+cn* en1 en1 CyO Vials with engrailed flies 50% 50% en1 CyO b pr enxcn en1

  49. Cytogenomics Salivary gland chromosome: high resolution cytogenetics.

  50. Cytogenomics Obsolete

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